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Introduction

Dynabeads® DNA DIRECT™ Blood is designed for the simple and rapid isolation of high quality PCR-ready genomic DNA from blood. This cost-effective and efficient procedure is completed in a single tube in only 15 minutes. DNA isolation involves cell lysis to release DNA and then adsorption of the DNA to the surface of the Dynabeads® . This is followed by magnetic separation of the intact DNA/Dynabeads® complex and subsequent washing to remove residual contaminants and potential PCR-inhibitors from the isolated DNA. After resuspension, the complex is ready for direct use in downstream PCR-reactions. The DNA may be eluted from the Dynabeads® by a short incubation at 65°C.

Dynabeads® DNA DIRECT™ Blood is ideal for DNA isolation from multiple samples and multiple PCR-analyses on each sample. The method can be automated.

Product Performance

Dynabeads® DNA DIRECT™ Blood allows reproducible isolation of genomic DNA as determined by PCR. The kit supports 100 isolations from 100 μl of blood. Each unit (200 μl) of Dynabeads® will isolate enough high quality genomic DNA for at least 100 PCR-amplifications. The protocol can also be modified to allow isolation from 500 μl blood samples, providing enough template- DNA for 1,000 PCR-amplifications.

Isolated DNA is free of RNA and contaminants that may inhibit PCR. The DNA is of high integrity and high molecular weight, and PCR-results show excellent reproducibility and high sensitivity. The procedure is simple and can be completed in as little as 15 minutes. Multiple samples may be handled in parallel and the isolation process can be automated.

The isolated genomic DNA depends on the number of nucleated cells present in the sample. From 100 μl blood the yield is typically 1-5 μg DNA. More DNA can be obtained with buffy coat as the starting sample.
Note: To isolate PCR-ready genomic DNA from small quantities of a wide variety of crude sample materials, use Dynabeads® DNA DIRECT™ Universal (Prod. No. 630.06).

Kit Components

Dynabeads® DNA DIRECT™ Blood is supplied complete with beads and the necessary buffers. All components are quality controlled and tested to be free of contaminating DNA.

Red Cell Lysis Buffer, supplied as a concentrate 1.6 M

Dynabeads® DNA DIRECT™ Blood, supplied ready to use in a lysis buffer; an aqueous suspension containing salts and detergent. The suspension is corrosive, as it contains 1-5 % NaOH.

Technical Tips

Sample Preparation

Fresh capillary and anticoagulated blood (EDTA, ACD, citrate and heparin) may be used directly in the protocols in the next section.

Mix blood stored at room temperature or 4°C on a vortex mixer for 2-3 seconds prior to aliquots being removed. Close the tube cap carefully before mixing. Take care when opening tubes to avoid creating aerosols of potentially infectious material.

Buffy coat contains 2-4 times the amount of white blood cells per volume compared to fresh blood. Only use 50 μl of buffy coat diluted with 50 μl PBS as starting material for the protocol.

Bone marrow samples also contain a high amount of DNA. Use 10-20 μl bone marrow as starting material following the protocol in the next section.

Note: Hypercellular samples (e.g. buffy coat, bone marrow and blood from individuals with acute infections) contain high numbers of nucleated cells and are thus richer in DNA than normocellular samples. This excess DNA may make the DNA/Dynabeads® complex harder to handle. In these cases, reduce the quantity of sample used in the procedure. Alternatively, increase the volume of Dynabeads® .

Choice of Magnet and Tubes.

Dynal® magnets pull the DNA/Dynabeads® complex to the tube wall so the supernatant can be removed. By scaling down the procedure it is possible to isolate genomic DNA from small samples in a 96 well plate using the Dynal® MPC™-96S (manually or automated).

Handling the DNA/Dynabeads® Complex

When adding Dynabeads® to the sample in a single rapid pipetting action, a complex of DNA and Dynabeads® will form. The complex will appear gelatinous. Do not mix further, as this will damage the DNA/Dynabeads® complex and reduce the yield of DNA isolated.

To avoid loss of material, do not share the complex until the Resuspension Buffer is added. To avoid withdrawing the complex into the pipette tip when aspirating the supernatant, a stepwise removal of the supernatant is recommended. Wash thoroughly to remove contaminants. Pipette the Washing Buffer into the test tube in a single, rapid pipetting action so that the DNA/Dynabeads® complex is swirled around in the buffer. The addition of Washing Buffer should wash the DNA/ Dynabeads® complex off the tube-wall in one piece. Some uncomplexed particles may be seen due to excess binding capacity.

Remove all Washing Buffer between each wash. After three washes, the DNA/Dynabeads® complex is broken up by resuspension in the Resuspension Buffer. Thoroughly resuspend by repeatedly pipetting the complex up and down until the complex has a homogeneous appearance. Do not stir using the pipette tip as DN may stick to the tip. Alternatively, move the tube rapidly over an uneven surface, e.g. an iron-thread tube rack, to achieve a shaking (not vortexing) force. Avoid pipetting air into the homogenised complex, as this may cause foaming.

PCR-Amplifications

Resuspended DNA/Dynabeads® can be used directly for PCR as the presence of Dynabeads® will not give any adverse effect. If the DNA is to be stored prior to PCR-amplification elution is recommended.

As one of the components in the kit contains NaOH, the isolated DNA is available for PCR-amplifications. To increase the specificity of primer annealing to the denatured DNA, do not place the PCR reaction-mixture on the thermal cycler until the temperature is well above annealing temperature.

Use 1% of the homogenized DNA/Dynabeads® complex in Resuspension Buffer as PCR-template. Up to a maximum of 5% of the complex or up to 50% of the eluted DNA can be used as starting material for PCR (50 μl volume).

Use PCR-profiles with 30-35 cycles.

Some PCR-reactions are very sensitive to the amount of template-DNA used. In such cases, perform a titration of the DNA/Dynabeads® complex and compare eluted with non-eluted DNA.

Note: For PCR-based tissue typing use 1/100 of the isolated DNA per PCR-reaction. This may be less template than is generally recommended, but due to the denatured state of the isolated DNA it will be more available for PCRamplifications. To increase the specificity of primer annealing to the denatured DNA, the PCR reaction-mixture on the thermal cycler until the temperature has reached 72°C.

Initial Checklist

During storage the Dynabeads® settle at the bottom of the bottle leaving a colorless, clear solution. Resuspend well by gentle shaking to obtain a homogeneous dispersion of beads in solution before use.

The Red Cell Lysis Buffer and the Washing Buffer are supplied as concentrates and should be diluted to their correct working concentration using sterile and PCR-grade water and equipment before use. Note that the exact volumes supplied in the bottles may vary. Also note that the working concentration of the Red Cell Lysis Buffer is specific for each of the two different protocols described in below (referred to as 1x the Red Cell Lysis Buffer). The working concentration of the Washing Buffer is 1:10 for both protocols (referred to as 1x Washing Buffer).

Bring the Dynabeads® , 1x Washing Buffer and Resuspension Buffer to room temperature before use. The Red Cell Lysis Buffer should be used cold.

Isolation Protocols

Please read above section "Technical Tips" before use.

DNA Isolation Protocol from 100 μl Blood

In a 2 ml round-bottomed microcentrifuge tube mix 100 μl anti-coagulated blood with 1 ml pre-diluted Red Cell Lysis Buffer (diluted 1:5 from the original concentration supplied, stored at 2-8°C and used cold). If less than 10 μl of blood is used as starting material, this Red Cell Lysis step can be omitted from the protocol.

Leave at room temperature with occasional shaking of the tube until the red cells have lysed and the solutio is clear. Red Cell Lysis should give a clear solution of bright red colour (lysis is usually complete in 5 minutes).

Centrifuge for 30 seconds at 13,000 rpm in a microcentrifuge to pellet the white blood cells. A small, white or light grey haemoglobin-free pellet of WBC should be visible. Remove and discard supernatant.

Immediately add 200 μl (1 unit) of Dynabeads to the pellet in a single rapid pipetting action to mix the components. Do not vortex or mix further.

Leave tube undisturbed at room temperature for 5 minutes.

Place tube on the magnet. Allow the DNA/Dynabeads complex to move to the tube wall after 1 minute carefully pipette off and discard supernatant. The DNA/Dynabeads complex will have a brown gelatinous appearance. During this step and subsequent washes, ensure that the complex is not disturbed or broken up.

Remove the tube from the magnetic field and wash the DNA/Dynabeads complex with 1 ml 1x Washing Buffer (brought to room temperature prior to use). The buffer should be added in a single, rapid pipetting action washing theNA Note: complex off the wall of the tube. Do not break up the complex as this will reduce the DNA yield.

Replace the magnetic field, let the DNA/Dynabeads complex move to the side of the tube and after 1 minute, or when supernatant has cleared, pipette off and discard the supernatant.

Repeat step 7-8 twice, each time ensuring that the supernatant is completely removed and the DNA/Dynabeads complex is intact.

Remove the tube from the magnetic field and thoroughly resuspend the DNA/Dynabeads complex in 200 μl Resuspension Buffer or a volume suitable for your PCRapplication. This suspension can be used directly for PCR-amplification.

If required, elute the DNA by incubation at 65°C for 5 minutes. Immediately place the tube in the magnet for 30 seconds or until the suspension has cleared, pipette off supernatant and transfer to a clean tube. Elution should give a clear, noncoloured, bead-free solution.

1% of the DNA/Dynabeads complex will be sufficient DNA-template for one 30-35 cycle PCR-reaction. Up to a maximum of 5% of the complex or up to 50% of eluted DNA can be used as starting material for PCR (50 μl volume).

If determination of DNA concentration by absorbance measurements at OD260 is required, the DNA will first have to be eluted off the beads. Ensure that there are no beads left in the solution as they will interfere with the spectrophotometrical readings in an unfavourable way. Eluted/non-eluted DNA from 100 μl of blood is sufficient for at least 100 PCR-reactions. The DNA/Dynabeads suspension after step 10 can be used directly for PCR-amplification. To prevent autocatalytic degradation if the DNA is to be stored at 4°C or -20°C elute the DNA a described in step 11.

Note: Before first use of the product, you are strongly advised to read the section "Technical Tips".

Initially use the protocol in its 100 μl format to become familiar with the procedure before upscaling. Due to the size of the white blood cell (WBC) pellet after centrifugation and the handling of the DNA/Dynabeads complex use 2 ml round-bottomed microcentrifuge vials when working with 500 μl of blood. In this specific protocol the Red Cell Lysis Buffer is used in two versions. These are made as follows:

Add 1 ml Red Cell Lysis Buffer B1, mix and invert tube several times until the red cells have lysed and the solution is clear.

Microcentrifuge for 10-30 seconds at 13,000 rpm to pellet WBC. Remove and discard the supernatant.

Vortex tube to resuspend WBC pellet, add 1 ml Red Cell Lysis Buffer B2 and mix. If lumps are observed, leave to settle for one minute and transfer supernatant to a new tube.

Microcentrifuge for 10-30 seconds at 13,000 rpm, remove and discard supernatant. A small, white or light grey, haemoglobin-free pellet of WBC should be visible.

Vortex the tube and add 200 μl (1 unit) of Dynabeads to the WBC pellet in a single rapid pipetting motion. Immediately aspirate the contents of the tube (using the same pipette tip) and transfer to a clean 2 ml tube. No incubation is necessary, but for maximum yield incubate for 5 minutes at room temperature. Do not vortex or mix further. At this point a complex of DNA/Dynabeads with a brown, gelatinous appearance should be visible.

Place the tube in the magnetic field and after 30-60 seconds, carefully aspirate and discard supernatant. During this step and subsequent washes, keep the complex intact.

Remove the magnetic field and add 1 ml 1x Washing Buffer. Use a single, rapid pipetting action to flush the complex from the tube wall. Do not break up the complex.

Replace the magnetic field and after 30 seconds, aspirate and discard the supernatant.

Wash the complex twice by repeating steps 8 and 9 ensuring the supernatant is completely removed and the complex is intact.

Remove the magnetic bar. Add 200 μl Resuspension Buffer and disrupt the complex by repeatedly pipetting up and down. This suspension can be used directly for PCR-amplification

If elution is required, incubate at 65°C for 5 minutes and place the tube directly in the magnetic field. After 1 minute, transfer supernatant with the DNA to a new tube. Elution should give a clear, bead-free solution.

10-20 μl of the isolated and eluted DNA can be used to prepare a working dilution sufficient for 100 x 10 μl PCR reactions. The remainder may be stored frozen.

If determination of DNA concentration by absorbance measurements at OD260 is required, the DNA will first have to be eluted off the beads. Ensure that there are no Dynabeads left in the solution as the beads will interfere with the spectrophotometrical readings.

The DNA/Dynabeads suspension after step 11 can be used directly for PCR-amplification. To prevent autocatalytic degradation if the DNA is to be stored at 4°C or -20°C, elute the DNA as described in step 12.

Note: If samples from patients with abnormal white cell counts are used, the DNA/Dynabeads complex might not form or be too large to handle. In these cases, the initial volume of blood used accordingly.